Obesity and cardiometabolic risk have been associated with vitamin D levels even in children. The objective of the present study was to evaluate the association between insulin resistance (IR), cardiometabolic risk factors, and vitamin D in children from prepubertal to pubertal stages. A total of 76 children from the PUBMEP study, aged 4–12 years at baseline, were included. Children were evaluated in prepubertal and pubertal stages. Anthropometric measurements and selected cardiometabolic risk biomarkers, such as plasma glucose, blood lipids, insulin, adiponectin, leptin, and blood pressure, and serum 25-hydroxyvitamin D (25(OH)D) were determined. Children were categorized by obesity degree and IR status combined before and after puberty. Paired t-test and multivariate linear regression analyses were conducted. During puberty, the increase in triacylglycerols, insulin, and HOMA-IR and the decrease in QUICKI were significantly associated with the reduction in 25(OH)D (B = −0.274, p = 0.032; B = −0.219, p = 0.019; B = −0.250, p = 0.013; B = 1.574, p = 0.013, respectively) after adjustment by BMI-z, sex, and pubertal stage. Otherwise, prepubertal non-IR children with overweight/obesity that became IR during puberty showed a significant decrease in 25(OH)D and HDL-c, and an increase in waist circumference and triacylglycerol concentrations (p < 0.05 for all) over time. These results suggest that changes in IR seem to be associated with an effect on 25(OH)D levels during puberty, especially in children with overweight.